Extracellular nucleotides may play important regulatory roles within the cardiovascular system (Kunapuli and Daniel, 1998; Vassort, 2001), with recent evidence implicating P2 purinergic receptors in protection of ischemic or reperfused myocardium (Vassort, 2001; Ninomiya et al., 2002a,b). Although purine nucleotides have been the most widely studied P2 agonists, it is clear pyrimidines such as UTP also possess important regulatory functions. Two recent studies provide evidence that UTP may mediate protective actions in ischemic hearts (Yitzhaki et al., 2006) and hypoxic myocytes (Yitzhaki et al., 2005) from rats. However, not all investigations detect such effects of UTP under similar conditions (Ninomiya et al., 2002b), and a number of issues arise from prior studies, foremost among these being the use of very high UTP concentrations to trigger protection (Ninomiya et al., 2002b; Yitzhaki et al., 2005). These prior studies use levels >2 orders of magnitude above those required to activate the P2Y2 receptors implicated in the responses (von Kügelgen and Wetter, 2000; Jacobson et al., 2002; von Kügelgen, 2006). The physiological (even pathological) relevance of responses to such high agonist levels is questionable, and it is difficult to ascribe effects to specific receptor subtypes, because such concentrations will even activate P2 receptors classified as UTP-insensitive (e.g., P2Y1 receptors). Furthermore, in the only study of intact perfused hearts (Ninomiya et al., 2002b) myocyte death was not assessed, and the primary measure of ischemic outcome (contractile recovery) was complicated by uncontrolled and thus variable heart rate. This renders interpretation of rate-dependent contractile function problematic, potentially explaining lack of effect of UTP in this work (Ninomiya et al., 2002b). Another unresolved issue relates to potential roles of endogenous P2 agonists. Whether ischemia enhances interstitial pyrimidine nucleotide levels sufficiently to activate P2 receptors is not known. Interestingly, recent work does reveal elevated vascular UTP in humans suffering myocardial ischemia (Wihlborg et al., 2006), providing indirect support for cardiac UTP release.

Four inter-related questions emerge regarding UTP and cardiac protection, to be addressed in part here: 1) do low and physiologically relevant concentrations of UTP enhance ischemic tolerance; 2) is observed protection with UTP dependent upon P2 receptor activation; 3) does ischemia trigger significant accumulation of UTP and other P2 agonists in cardiac interstitium; and 4) are these interstitial agonist concentrations sufficient to activate P2 receptors and so modify ischemic tolerance? We tested the ability of 250 nM UTP to modify functional outcomes and cell death following ischemia-reperfusion, assessed the impact of P2 antagonism on UTP responses, characterized the impact of ischemia on cardiac interstitial [UTP], and tested for protection in response to intrinsic P2 agonism [through P2 antagonism with suramin, RB-2, PPADS, and pyridoxal-α5-phosphate-6-phenylazo-4′-carboxylic acid (MRS2159)].

Materials and Methods

Animals. Experiments were performed in accordance with the Institute of Laboratory Animal Resources (1996) and work approved by the Institutional Animal Care and Use Committee. Young male C57/Bl6 mice (16–20 weeks of age) were used in the studies. All animals were allowed at least 4 days of in-house acclimatization before experimental procedures.

Chemicals and Reagents. All drugs used were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Drug solutions were infused into hearts at ≤1% of total coronary flow rate to achieve the final concentrations indicated.

For assessment of necrotic cell death, we measured efflux of the intracellular enzyme LDH (Headrick et al., 2001; Peart and Headrick, 2003; Reichelt et al., 2005). Myocardial LDH efflux has been shown to correlate linearly with infarct size in this model (Peart and Headrick, 2003). Coronary effluent was collected on ice, and it was stored at–80°C (≤7 days) until analysis for LDH via a CytoTox assay kit (Promega Corporation, Annandale, NSW, Australia), a colorimetric assay coupling LDH content to conversion of a tetrazolium salt to a colored formazan product. Total LDH washed from hearts during reperfusion was calculated as the product of LDH content (per milliliter) and total coronary effluent over the measurement period (Headrick et al., 2001; Peart and Headrick, 2003).

Cardiac Microdialysis Assessment of Interstitial Nucleotides. To test for elevations in endogenous P2 agonists in the interstitial compartment, a second group of untreated hearts (n = 14) was instrumented with cardiac microdialysis probes according to the method outlined by us previously (Peart and Headrick, 2000). Probes were constantly perfused at 1 μl/min, and eluents were collected on ice over 10-min periods. After a 60-min stabilization period half of these hearts were subjected to 20-min global ischemia and 45-min reperfusion, whereas the remainder were perfused normally (nonischemic control). Consecutive 10-min samples of dialysate were collected, and the dialysate was frozen at–80°C until analyzed via high-performance liquid chromatography, using an adaptation of prior techniques (Headrick and Willis, 1989; Harrison et al., 1998). In brief, dialysate samples were injected onto a Suplecosil C18S column (maintained at 27°C) and eluted at 1 ml/min using a buffer gradient (60% solution A/40% solution B at 0 min to 40% solution A/60% solution B at 25 min, followed by isocratic elution to 60 min). Solution A consisted of a 10 mM KH2PO4 buffer containing 0.25% MeOH and 10 mM tetrabutylammonium hydroxide, pH 6.9. Solution B consisted of a 50 mM KH2PO4 buffer containing 30% MeOH and 5.3 mM tetrabutylammonium hydroxide, pH 7.0. Eluent absorbance was continuously monitored on a Waters photodiode array detector (Waters, Sydney, NSW, Australia), and nucleotide concentrations were calculated based on comparison with absorbances for known standards run each day of analysis.

Statistical Analysis. Data are presented as mean ± S.E.M. Baseline data and functional outcomes from ischemia in different experimental groups were compared via one-way analysis of variance. When significant effects were identified a Newman-Keuls post hoc test was used for individual comparisons. A value of P < 0.05 was considered significant in all tests.

Results

Normoxic Function. Baseline functional data are provided in Table 1. Pressure development and inotropic and lusitropic states were high, and coronary flow was submaximal, based on prior data for flow responses in murine hearts (Headrick et al., 2001). Treatment with UTP failed to modify ventricular contractile function in normoxic hearts. Likewise, most antagonist treatments did not alter contractility, although 100 μM (but not 10 μM) RB-2 did generate a significant increase in force development and inotropic state (Table 1). Interestingly, suramin (at 200 μM) and RB-2 (at both 10 and 100 μM) generated significant elevations in baseline coronary flow, whereas there was a tendency (albeit insignificant) for modestly decreased coronary flow with UTP (Table 1).

Data were measured immediately before ischemia. All values are means ± S.E.M.

Effects of Ischemia and UTP on Cardiovascular Function and Cell Death. Ischemia resulted in sustained depression of contractile function over the 45-min reperfusion period (Fig. 1). Diastolic pressure remained elevated at ∼20 mm Hg (Fig. 1A), and ventricular pressure development recovered to ∼65% of preischemic levels (Fig. 1B). Coronary flow recovered to slightly less than preischemic levels (Fig. 1C). Ischemia-reperfusion was also associated with significant efflux of LDH (Fig. 2), reflecting necrotic death (Peart and Headrick, 2003).

Treatment of hearts with 250 nM UTP significantly reduced postischemic diastolic dysfunction, and it moderately enhanced ventricular pressure development (Fig. 1). These effects were not associated with significant differences in final recovery of flow rate (expressed as milliliters per minute per gram; Fig. 1C). Treatment with UTP also significantly reduced LDH loss (Fig. 2). We additionally tested for effects of the P2Y1 agonist 2-MeSATP at 50 nM, but the agent was found to exert no effects on any markers of ischemic injury at this concentration (data not shown). To test for P2 involvement in UTP-mediated cardioprotection, we cotreated hearts with the antagonist suramin, which was found to exert concentration-dependent effects on ischemic outcome. When applied at 200 μM, suramin worsened functional recovery from ischemia (Fig. 1) and LDH washout (Fig. 2). However, a lower 10 μM concentration was without effect on functional recovery from ischemia (Fig. 1). Importantly, UTP was unable to modify responses to ischemia in the presence of 10 or 200 μM suramin, failing to reduce contractile dysfunction (Fig. 1, A and B) below levels observed in the presence of suramin alone. Likewise, UTP no longer reduced LDH washout in the presence of 200 μM suramin (Fig. 2).

Effects of Ischemia on Myocardial Interstitial UTP, ATP, and ADP Concentrations. Based on apparent reduction in ischemic tolerance with 100 μM suramin alone, we assessed release of P2 agonists into the interstitial compartment (Fig. 3). Under normoxic conditions, we were able to detect ATP in most cases; yet, we could not consistently detect ADP or UTP in cardiac microdialysate samples (Fig. 3). However, during 20 min of global ischemia the accumulation of all three nucleotides was significantly enhanced, with ATP levels exceeding those for ADP, and in turn UTP. Nucleotide concentrations peaked within the initial 10 min of ischemia and then declined during the ensuing 10 min (although remaining substantially elevated above normoxic levels). Subsequent reperfusion led to a gradual decline in dialysate UTP, ATP, and ADP levels, such that only ATP remained detectable during the final 20 min of reperfusion. No changes in nucleotide levels were detected in normoxically perfused hearts (Fig. 3).

Discussion

Recent studies provide good support for UTP-dependent cardioprotection in the rat (Yitzhaki et al., 2005, 2006). However, these findings are not universal (e.g., Ninomiya et al., 2002b), and a number of interesting questions arise from these prior investigations. Here, we confirm P2-mediated cardioprotection in response to submicromolar concentrations of UTP, we document significant accumulation of UTP (together with ATP and ADP) in the interstitium of ischemic hearts, and we show that high levels of the P2 antagonists suramin and RB-2 (and not PPADS or MRS2159) worsen ischemic outcome. Interestingly, although a lower 10 μM concentration of RB-2 also limited postischemic outcome, suramin at this concentration was ineffective. Collectively, these findings evidence cardioprotection via exogenous UTP (involving limitation of ventricular contractile dysfunction and necrosis), and they implicate the P2Y2 receptor as the most likely mediator of this protection. Mixed effects of P2 antagonists alone also support protection via endogenous P2 agonists, although these responses may potentially involve P2-independent effects and they deserve further attention (see below).

P2 Purinoceptors and Cardioprotection. Based on structural similarities the P2Y receptor family is grouped into P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors, and into a dissimilar group containing P2Y12 and P2Y13 receptors (Kunapuli and Daniel, 1998; von Kügelgen and Wetter, 2000; Vassort, 2001; Jacobson et al., 2002). These G-coupled receptors, or their mRNAs, have been localized to cardiomyocytes (Webb et al., 1996), cardiac fibroblasts (Zheng et al., 1998), and vascular smooth muscle and endothelium (Wang et al., 2002), and they are known to regulate the activity of multiple intracellular kinases (von Kügelgen and Wetter, 2000) to modify cellular function. Relatively little work has been undertaken to delineate functions of P2Y purinoceptors in myocardial stress resistance and protection. Unfortunately, delineating contributions of individual P2 subtypes to functional responses in intact tissue is hampered by mixed selectivities of agonists and particularly antagonists (von Kügelgen and Wetter, 2000; Jacobson et al., 2002). Although judicious choice of drug concentration can limit the range of receptor candidates, recent studies of cardiac protection have used very high (3–100 μM) UTP concentrations (Ninomiya et al., 2002b; Yitzhaki et al., 2005), sufficient to maximally activate P2Y2, P2Y4, and P2Y6 receptors, and potentially activate P2Y1 and P2Y11 receptors at which UTP is generally considered a poor agonist (von Kügelgen and Wetter, 2000; Jacobson et al., 2002). Here, we use a low UTP concentration of 250 nM that, based on known potencies (Jacobson et al., 2002), should be selective for P2Y2 receptors but may also activate P2Y4 receptors to a limited extent. Because UTP possesses EC50 values at P2Y1, P2Y6, and P2Y11 receptors as much as 3 orders of magnitude higher than the concentration applied here, these receptors should not be significantly activated. Furthermore, we observed no effects of P2Y1 agonism with 2-MeSATP (see Results). Finally, the 10 and 200 μM concentrations of the nonselective P2Y antagonist suramin shown to inhibit UTP-mediated protection (Fig. 1) should exert greatest effects at P2Y2, P2Y1 (eliminated as a candidate due to lack of effect of 2-MeSATP), and P2Y11 receptors (eliminated as a candidate because of their insensitivity to 250 nM UTP). Collectively, the effects of 250 nM UTP and suramin, and lack of effect of 2-MeSATP, implicate P2Y2 receptor involvement in UTP-dependent cardioprotection.

In contrast to our data, a prior study in rats found that myocardial P2Y activation alone does not generate protection, although endogenous P2 agonists apparently contributed to benefit with ischemic preconditioning (Ninomiya et al., 2002a,b). Limitation of ischemic injury observed here (Figs. 1 and 2) is consistent with UTP-mediated protection of hypoxic cardiomyocytes (Yitzhaki et al., 2005). Furthermore, in the course of the current study, Yitzhaki et al. (2006) published details of infarct-sparing effects of transient UTP pretreatment in an in vivo rat model. These investigators found UTP reduced hypoxia-dependent mitochondrial Ca2+ load in isolated myocytes, potentially underlying observed protection. Reasons for the lack of effect of UTP in the earlier study of Ninomiya et al. (2002b) versus protection observed here and by Yitzhaki et al. (2006) are not known, although high UTP levels used in prior studies raise the possibility of confounding effects of multiple P2Y subtype activation. Moreover, Ninomiya et al. (2002b) did not assess markers of cell death, and heart rate was uncontrolled, complicating interpretation of rate-dependent contractility.

Ischemic Accumulation of Interstitial Nucleotides. Cardiac microdialysis provides a useful tool for monitoring accumulation of signaling compounds in the myocardial interstitium (Harrison et al., 1998; Peart and Headrick, 2000; Ninomiya et al., 2002a). Although some degree of tissue injury is unavoidable in microdialysis studies, and the injury is perhaps exaggerated in small technically challenging models such as the mouse (Peart and Headrick, 2000), our data nonetheless reveal substantial ischemia-specific elevations in UTP, ATP, and ADP in cardiac microdialysate (Fig. 3). Although prior studies have documented similar elevations in ATP and ADP in rat myocardium (Ninomiya et al., 2002a), together with elevations in the related P1 agonist adenosine in rat and mouse hearts (Harrison et al., 1998; Peart and Headrick, 2000), this is the first report of ischemic elevations in myocardial interstitial UTP. Recent observations of vascular accumulation of UTP in patients suffering myocardial ischemia are consistent with cardiac UTP release (Wihlborg et al., 2006).

The source and identity of nucleotides released during ischemia remains unclear: nucleotides may be released from endothelium by G-coupled receptor activation (Yang et al., 1994), from sympathetic and parasympathetic nerve endings (Burnstock and Kennedy, 1986), and from cardiac myocytes during hypoxic or ischemic stress (Forrester and Williams, 1977; Clemens and Forrester, 1980). The changes in interstitial nucleotides observed here could play a regulatory role during or following ischemia: interstitial UTP, ATP, or ADP may all activate G-coupled P2Y receptors (with ATP and ADP additionally modulating ion channel-coupled P2X purinoceptors) in coronary vessels (Strøbaek et al., 1996; Wang et al., 2002), cardiac fibroblasts (Zheng et al., 1998), and myocytes (Webb et al., 1996). Although it is problematic to estimate precise solute concentrations from microdialysate levels, our data suggest that low micromolar levels of UTP and ATP may be achieved with severe ischemic stress, sufficient to activate P2Y2 receptors at which these nucleotides possess EC50 values ≤0.25 μM (Jacobson et al., 2002).

Modulation of Ischemic Tolerance by P2 Antagonism. Having established that low levels of exogenous UTP trigger P2-dependent cardioprotection (Figs. 1 and 2) and that ischemia generates substantial elevations in this and other P2 agonists (Fig. 3), we sought to identify effects of endogenous nucleotides via cotreatment with differing P2 antagonists (Fig. 4). Suramin, RB-2, and PPADS are relatively broad-spectrum antagonists, with RB-2 exhibiting some selectivity for P2Y versus. P2X, whereas PPADS is partially selective for P2X versus P2Y receptors (Jacobson et al., 2002; von Kügelgen, 2006). The drug MRS2159 is selective at P2X1 receptors. It is worth noting that suramin and PPADS can exert effects on signal transduction and G proteins (Shehnaz et al., 2000). However, the polar nature of the agents prevents them from crossing the cell membrane, minimizing these effects in intact tissue.

Detrimental effects of 200 μM suramin and 10 to 100 μM RB-2 on postischemic recovery (Figs. 1 and 4), together with lack of effect of PPADS and MRS2159, tend to support intrinsic activation of P2Y purinoceptors. Since suramin selectively blocks P2Y2 versus P2Y4 receptors (at which it exhibits very poor potency) (von Kügelgen and Wetter, 2000; von Kügelgen, 2006), inhibitory effects of this agent on both UTP-mediated protection and intrinsic ischemic tolerance support involvement of P2Y2 receptors. Lack of effect of PPADS on ischemic outcome is also consistent with P2Y2 involvement, because PPADS is most potent at P2Y1 and possibly P2Y6 receptors, but it is ineffective at rodent P2Y4 and human P2Y2 receptors (von Kügelgen and Wetter, 2000; von Kügelgen, 2006). In contrast, RB-2 will more broadly antagonize P2Y receptor subtypes, and it is also shown to affect on postischemic recovery at low and high concentrations (Fig. 4). However, a complication in this interpretation relates to potential blockade of ectonucleotidase activity by suramin and other P2 antagonists.

Several studies confirm that different P2 antagonists may noncompetitively inhibit ectonucleotidase activity in different species and tissues (Chen et al., 1996; Yegutkin and Burnstock, 2000). Studying Mg2+-sensitive ectonucleotidase from rats, Yegutkin and Burnstock (2000) arrived at an IC50 of 20 to 30 μM for suramin (with values of 50 μM for RB-2 and ∼1 mM for PPADS). Chen et al. (1996) acquired IC50 values of 40 to >100 μM for suramin in rat and mouse cells (and 15–20 μM for both RB-2 and PPADS). These inhibitory effects will limit responses mediated by hydrolysis products of extracellular nucleotides, which may be responsible (at least in part) for the detrimental actions of these agents during ischemia-reperfusion. In assessing this possibility, we examined responses to lower 10 μM concentrations of suramin and RB-2 (Figs. 1 and 4), which we reasoned should retain some P2 inhibitory efficacy but be less effective in inhibiting ectonucleotidases. It is interesting that, although the lower RB-2 concentration still effectively impaired postischemic outcome, 10 μM suramin was without effect on the response to ischemia (Fig. 4). This likely stems from differing inhibitory potencies of the two agents at different P2 receptors (see above), but it may additionally reflect effects of suramin on other processes dictating postischemic outcome (including inhibition of ectonucleotidase activity) at high concentrations. In this regard, preliminary findings of Eckle et al. (2006) reveal that gene ablation of CD73 worsens outcome from ischemia in vivo, and we have recently confirmed that P1 receptor activation by endogenous adenosine (which is generated via CD73 activity) does limit injury during ischemia-reperfusion (Reichelt et al., 2005). We must temper our conclusion then, that effects of the P2 antagonist suramin may involve a combination of receptor antagonism and/or inhibition of beneficial actions of ectonucleotidase activity. Future work might address the impact of these agents on interstitial accumulation of nucleotides and their hydrolysis products.

Injurious effects of suramin and RB-2 in ischemic myocardium have not been documented previously. The studies of Ninomiya et al. (2002a,b) in rat hearts do not report on effects of P2 antagonism alone, and in any case they use an index of postischemic outcome that is difficult to interpret (rate-dependent contractile function in unpaced hearts). However, other evidence does support endogenous P2 receptor activation or roles for nucleotides in ischemic myocardium: cardiac tissue plasminogen activator release during ischemia is modulated by endogenous nucleotides acting at P2Y receptors (Olivecrona et al., 2007); intrinsic P2 receptor activation contributes to preconditioning with transient ischemia (Ninomiya et al., 2002a); and as already noted, CD73 knockout may worsen ischemic tolerance (Eckle et al., 2006), supporting a contribution of extracellular nucleotide hydrolysis to cardiac protection. Thus, there is evidence for roles of endogenous nucleotides in ischemic myocardium.

P2 Purinoceptor-Dependent Coronary Constriction. Unexpected coronary dilation in response to suramin and RB-2 is of interest because both antagonists significantly enhanced coronary flow in normoxic hearts (Table 1). Additionally, and perhaps related, the P2 agonist UTP elicited a small (albeit insignificant) decline in baseline flow. There is some prior support for P2Y-mediated smooth muscle constriction, in addition to dilation (Corr and Burnstock, 1991; Murthy and Makhlouf, 1998), although substantial species differences exist in these responses. This may explain the modest dilatory effect of extremely high (50 μM) UTP reported by Ninomiya et al. (2002b) in rat hearts versus modest (insignificant) constriction observed with UTP in the mouse (Table 1). Other studies report on novel UTP-sensitive coronary P2Y receptors exhibiting some properties relevant to the current observations (Hill and Sturek, 2002), and they support UTP-mediated coronary constriction (Matsumoto et al., 1997). Rayment et al. (2007) most recently presented evidence for P2Y2-dependent UTP-mediated coronary artery contraction, which is sensitive to suramin. Thus, despite P2-mediated endothelium-dependent dilation of intact vessels (Vials and Burnstock, 1994; Marrelli, 2001), evidence also reveals a P2Y-mediated coronary constriction (Matsumoto et al., 1997; Sugimura et al., 2000). The nature of these different vascular responses deserves further attention, as does the identity of P2 receptors involved. For example, although evidence is presented for P2Y2 involvement in coronary constriction (Rayment et al., 2007), P2Y6-dependent contraction detected in other vessel types (Malmsjö et al., 2003) could conceivably play some role.

Conclusions. This study builds on prior evidence of UTP-mediated tissue protection (Yitzhaki et al., 2005, 2006). Our observations reveal that low concentrations of UTP improve postischemic outcomes in a P2-dependent manner. Although the receptor subtype(s) involved remains to be unequivocally identified, the P2Y2 subtype is most strongly implicated based on the effects of UTP and suramin. Ischemia itself also triggers elevations in interstitial UTP and other P2 agonists (ATP and ADP) to active levels, and P2 antagonism with suramin and RB-2 does limit intrinsic ischemic tolerance. Concentration-dependent actions of suramin warrant further study, and they may reflect differing potency at relevant P2 receptors and/or other inhibitory actions (such as blockade of ectonucleotidase activity).

Footnotes

This work was supported by grants from the National Health and Medical Research Council of Australia and the National Heart Foundation of Australia. We gratefully acknowledge scholarship support for S.W. (Australian Postgraduate Award), and fellowship support for J.P.H. (National Heart Foundation of Australia) and J.N.P. (National Health and Medical Research Council of Australia).

Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of Laboratory Animals 7th ed. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, Washington DC.